Abstract
The RNA-binding protein PKR serves as a crucial antiviral innate immune factor that globally suppresses translation by sensing viral double-stranded RNA (dsRNA) and by phosphorylating the translation initiation factor eIF2α. Recent findings have unveiled that single-stranded RNAs (ssRNAs), including in vitro transcribed (IVT) mRNA, can also bind to and activate PKR. However, the precise mechanism underlying PKR activation by ssRNAs, remains incompletely understood. Here, we developed a NanoLuc Binary Technology (NanoBiT)-based in vitro PKR dimerization assay to assess the impact of ssRNAs on PKR dimerization. Our findings demonstrate that, akin to double-stranded polyinosinic:polycytidylic acid (polyIC), an encephalomyocarditis virus (EMCV) RNA, as well as NanoLuc luciferase (Nluc) mRNA, can induce PKR dimerization. Conversely, homopolymeric RNA lacking secondary structure fails to promote PKR dimerization, underscoring the significance of secondary structure in this process. Furthermore, adenovirus VA RNA 1, another ssRNA, impedes PKR dimerization by competing with Nluc mRNA. Additionally, we observed structured ssRNAs capable of forming G-quadruplexes induce PKR dimerization. Collectively, our results indicate that ssRNAs have the ability to either induce or inhibit PKR dimerization, thus representing potential targets for the development of antiviral and anti-inflammatory agents.